CN114563567A - 一种检测LChV-1病毒的胶体金试纸条及其制备方法 - Google Patents
一种检测LChV-1病毒的胶体金试纸条及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种检测LChV‑1病毒的胶体金试纸条及其制备方法,属于大樱桃病毒病检测领域。所述胶体金试纸条包括PVC板,依次搭接有样品垫,结合垫,硝酸纤维素膜和吸水纸;所述结合垫是结合有胶体金颗粒标记的兔LChV‑1病毒多克隆抗体的玻璃纤维素膜;所述硝酸纤维素膜上依据样品流动方向依次设置有检测线和质检线,所述检测线包被有LChV‑1病毒重组蛋白,所述质检线包被羊抗兔IgG抗体。本发明制备的胶体金试纸条在田间可以快速检测LChV‑1病毒,且特异性好,灵敏度高。
Description
技术领域
本发明涉及一种检测LChV-1病毒的胶体金试纸条及其制备方法,属于大樱桃病毒病检测领域。
背景技术
病毒检测的方法主要有,血清学检测、电子显微镜观察、指示植物接种、通过PCR或RT-PCR进行的DNA扩增、芯片杂交等。以上方法均需要实验室条件下完成,不能满足田间快速检测LChV-1的要求。
甜樱桃生产在改善农村经济状况以及农业产业化方面发挥着非常重要的作用。我国樱桃主产区主要有山东、辽南地区、冀东地区、陕西、北京和河南,山东是我国甜樱桃栽培面积最大、产量最多的一个省。然而,甜樱桃病毒病的发生却严重影响樱桃产量和品质,是制约樱桃产业持续发展的重要因素之一。目前,国内已报道侵染甜樱桃的病毒病有14种,其中樱桃小果病毒1(Little cherry virus 1,LChV-1)在我国大部分樱桃产区均有分布,侵染后可引起果实变小、甜度降低、晚熟等症状,严重影响了樱桃产量,带来很大的经济损失。LChV-1是长线形病毒科长线病毒属,2004年波兰首次报道发现LChV-1,我国于2015年首次报道LChV-1侵染甜樱桃,该病毒主要通过汁液和昆虫介体等途径传播。针对樱桃小果病毒1的检测,急需一种快速的,低成本的检测方法。
发明内容
本发明针对上述问题,提供了一种检测LChV-1的胶体金试纸条及其制备方法。本发明采用胶体金试纸条检测LChV-1病毒,检测时间短,成本低,结果容易判断,可用于田间快速检测。本发明的技术方案如下:
一种检测LChV-1病毒的胶体金试纸条,包括PVC板,依次搭接有样品垫,结合垫,硝酸纤维素膜和吸水纸;
所述结合垫是结合有胶体金颗粒标记的兔LChV-1病毒多克隆抗体的玻璃纤维素膜;
所述硝酸纤维素膜上依据样品流动方向依次设置有检测线和质检线,所述检测线包被有LChV-1病毒重组蛋白,所述质检线包被羊抗兔IgG抗体。
进一步的,所述LChV-1病毒重组蛋白核苷酸序列如SEQ No.1所示;所述LChV-1病毒重组蛋白的制备方法,步骤如下:
设计带有酶切位点的特异性引物用于构建原核表达载体,引物序列如下:
LChV-1-CP-BamHI-12061-F:
AAGGATCCATGGCGAATTTAGCTTTCACGAAA;
LChV-1-CP-NotⅠ-13275-R:
TTGCGGCCGCTTAATTTCTTCTACCGCGACGTGG;
成功构建PE-HIS-LChV-1-CP原核表达载体;将PE-HIS-LChV-1-CP转入大肠杆菌表达株系的Rosetta细胞中,加IPTG诱导表达获得LChV-1病毒CP的重组蛋白;并过镍柱纯化,经得到纯化后的LChV-1病毒重组蛋白;优选的,所述IPTG的浓度为0.5mmol/L。
进一步的,利用纯化后的LChV-1病毒重组蛋白通过常规方法制备兔LChV-1病毒多克隆抗体。
进一步的,胶体金颗粒标记的兔LChV-1病毒多克隆抗体(金标抗体)的制备,步骤如下:
调节胶体金溶液pH至7.2,将兔LChV-1病毒多克隆抗体加入胶体金溶液中,其中兔LChV-1病毒多克隆抗体的终浓度为0.24mg/μL;涡旋震荡,室温放置1h后加入终浓度为1%的BSA震荡,室温静置1h;13000rpm,4℃离心25min,弃上清;加浓度为2%的BSA(其中含0.01mol/L sodium borate)重悬沉淀;13000rpm,4℃离心25min,弃上清;加TBS重悬沉淀,获得胶体金颗粒标记的兔LChV-1病毒多克隆抗体。
优选的,所述胶体金溶液中胶体金颗粒的粒径为40nm;胶体金颗粒标记的兔LChV-1病毒多克隆抗体中多克隆抗体的浓度为2.5μL/cm。
进一步的,本发明检测LChV-1病毒的胶体金试纸条的制备方法,步骤如下:
(1)将上述制备方法获得的胶体金颗粒标记的兔LChV-1病毒多克隆抗体滴加到玻璃纤维素膜上,滴加至饱和状态,室温晾干,得到结合垫;
(2)将LChV-1病毒重组蛋白和羊抗兔IgG抗体分别点涂于硝酸纤维素膜上,得到含有检测线和质检线的硝酸纤维素膜;优选的,所述检测线上重组蛋白浓度为0.24mg/ml,羊抗兔IgG抗体浓度为0.04mg/ml;
(3)在PVC板上沿样品流动方向依次搭接样品垫,结合垫,硝酸纤维素膜和吸水纸,组装得到胶体金试纸条。
本发明与现有技术相比具有以下优点:
本发明制备的胶体金试纸条在田间可以快速检测LChV-1病毒,且特异性好,灵敏度高。
附图说明
图1为LChV-1病毒多克隆抗体标记最佳浓度测定结果图;其中,0,0.24,0.48,0.72,0.96为LChV-1多克隆抗体终浓度mg/ml;
图2为试纸条检测线LChV-1病毒重组蛋白最佳浓度测定图;其中,2、1、0.5、0.25、0.125、0.0625为LChV-1多克隆抗体终浓度mg/ml;
图3为试纸条质检线羊抗兔IgG抗体最佳浓度测定图;其中,0.1、0.08、0.06、0.04、0.02为羊抗兔IgG抗体终浓度mg/ml;
图4为胶体金试纸条结构图;
图5为胶体金试纸条判定结果图;
图6为病毒样本检测结果图;
图7为田间样本检测结果图;
图8为田间样品RT-PCR检测结果图。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1:LChV-1病毒重组蛋白及制备方法
设计带有酶切位点的特异性引物用于构建原核表达载体,引物序列如下:
LChV-1-CP-BamHI-12061-F:
AAGGATCCATGGCGAATTTAGCTTTCACGAAA;
LChV-1-CP-NotⅠ-13275-R:
TTGCGGCCGCTTAATTTCTTCTACCGCGACGTGG;
成功构建PE-HIS-LChV-1-CP原核表达载体;将PE-HIS-LChV-1-CP转入大肠杆菌表达株系的Rosetta细胞中,加浓度为0.5mM IPTG诱导表达获得LChV-1病毒CP的重组蛋白;并过镍柱纯化,经得到纯化后的LChV-1病毒重组蛋白。
实施例2:LChV-1病毒多克隆抗体及制备方法
将实施例1获得的LChV-1病毒重组蛋白注射新西兰大白兔,初次免疫量为400μg蛋白;以后进行三次加强免疫,每次免疫量为200μg蛋白。耳静脉取血,ELISA测定免疫效价。达到免疫标准后,进行采血收取血清,得到LChV-1病毒CP蛋白的重组蛋白的兔多克隆抗体,于-20℃保存。
实施例3:胶体金颗粒标记的LChV-1病毒多克隆抗体(金标抗体)的制备
将不同终浓度(0,0.24,0.48,0.72,0.96mg/ml)的抗血清加入离心管中,加入pH为7.2粒径大小为40nm的胶体金溶液(用0.2mol/L碳酸钾调节pH)。5min后加入终浓度为10%NaCl,持续1h后观查颜色变化,确定颜色不变的最低浓度即为最佳抗体标记浓度,故确定最佳标记浓度为0.24mg/ml(图1所示)。
金标抗体制备步骤如下,取1mL上述胶体金溶液,加入抗血清,迅速震荡5min,室温静置1h。加入BSA至终浓度为1%,震荡5min后静置1h,13000rpm 4℃离心25min,小心弃上清。加入1ml 2%BSA含0.01mol/Lsodium borate重悬沉淀,13000rpm 4℃离心25min,小心弃上清。加入200ul TBS重悬沉淀,得到金标抗体。
实施例4:LChV-1病毒的胶体金试纸条制备方法及专一性检测
将实施例3获得金标抗体涂在已经封闭好的玻璃纤维素膜上,室温晾干;将不同终浓度(0.0625,0.125,0.25,0.5,1mg/ml)重组蛋白点涂于硝酸纤维素膜上,做为检测线,如图2所示,肉眼可识别的最低浓度即为最佳检测线浓度,最终确定最佳检测线浓度为0.25mg/mL。将不同终浓度(0.1,0.08,0.06,0.04,0.02mg/ml)羊抗兔IgG抗体点涂于硝酸纤维素膜上,做为质检线,如图3所示,肉眼可识别的最低浓度即为最佳质检线浓度,最终确定最佳质检线浓度为0.04mg/ml。点涂之后室温干燥,作为胶体金试纸条的显色部分。将硝酸纤维素膜贴于PVC底板的正中间,样品流动方向的上方搭接有吸水纸,滴加样品的方向搭接有样品垫和金标垫,搭接方式如图4所示。
试验例:
将植物样本用TBST缓冲液研磨后,滴加于胶体金试纸条上,几分钟即可显示结果,通过观察检测线和质检线的显色情况,即可判断所检测植物样本中是否有LChV-1病毒;结果判定如图5所示:
阳性:检测线没有红色条带,质检线出现红色条带,判定为阳性,说明所检测植物样本中有LChV-1病毒。
阴性:检测线和质检线两条线都显现红色条带,判定为阴性,即所检测样品中不含有LChV-1病毒。
无效:只有检测线有条带,或检测线质检线均没有条带,表明该试纸条检测结果不可信。
试验例1:特异性检测
用实验室现存单独侵染CMV(黄瓜花叶病毒),TYLCV(番茄黄叶曲叶病毒),PDV(李矮缩病毒)的植物样品进行检测。结果显示(图6),检测线不显色,质检线显色,说明该试纸条可以特异性检测LChV-1病毒。其余三种病毒检测显色,检测线显色,说明该试纸条特异性好,未出现假阳性。本发明试纸条可以特异性的检测出含有LChV-1病毒的植物样本。
试验例2:田间样本的检测
对所采集的大量田间样本检测,样本采集于烟台栖霞,采集时间为2021年7月份,品种为辉煌,玲珑脆,橹樱三,瑞德,五月红。结果显示(图7),样本1中检测出LChV-1,检测线不显色,质检线显色;样本2、样本3、样本4、样本5、样本6、样本7、样本8、样本9和样本10未检测出LChV-1,检测线和质检线均显色;将所采集的田间样品提取RNA进行RT-PCR检测,结果与试纸条结果一致。
检测步骤如下:提取植物叶片总RNA(TransZol Plant试剂提取法)利用RT-PCR进行核酸水平的病毒检测。RT反应体系(25.0μl)(试剂购自TakaRa公司):植物总RNA,20~50ng,反向引物(100mM)(引物序列参考专利ZL201811480617.2),5.0μl,10mM dNTP,4.0μl,dd H2O,5.0μl;80℃,3min,冰上5min;5×Reverse Transcriptase M-MLV Buffer,5.0μl,Reverse Transcriptase M-MLV,0.5μl,Recombinant RNase Inhibitor,0.5μl;42℃,90min。PCR体系(20.0μl):RT产物,2.0μl,正向引物,0.5μl,反向引物,0.5μl,2×TagMaster Mix(康为公司),10μl,dd H2O,7.0μl。反应条件:94℃,5min,94℃,40s,50~54℃,40s,72℃,20~35s,30Cycles,72℃,10min。
检测结果表明:本发明的田间应用具有良好的适应性,检测结果如图7所示。RT-PCR分子检测结果如图8所示。试纸条检测结果对1号样本检出LChV-1病毒,其余样本均未检测出,结果与RT-PCR检测结果一致。因此,在田间样品的检测中,本发明制备的胶体金试纸条可以成功检测出LChV-1病毒,具有良好的田间应用效果。
<110> 青岛农业大学
<120> 一种检测LChV-1病毒的胶体金试纸条及其制备方法
<130> 2022-1-0004
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1215
<212> DNA
<213> LChV-1病毒(Little cherry virus 1)
<400> 1
atggcgaatt tagctttcac gaaaaccttt gagagattga agcaattgct tccaatcgtt 60
caacctatat ggtcaattcc caatgctaat aggagtacgc agcaaaaatc tgacgttggg 120
gagtttgaat ccgcaatacg atctctgata aatgcacatg ctatctataa taaagaagat 180
ttgaataggg tttgtgagtt gggcgtaggt acaaatgctc ctgaagaagt taagaaactc 240
ttggtagatt taagaaattg cagaggaaag gcagacatga gttattttga agtggtaaaa 300
aattttagag agtacgtgag attaattgcc aaacctgaat cagacagaag tactgaagaa 360
actagtttga tcaataaatt tgaagaagcc ttgaaacttc tgacaaacaa aaaatcgact 420
ctgactaatg atgaacttaa atacttaaat catttcaata tacaagatat ccctgaacaa 480
cagcaaattc aatttaaaga tattttaaaa gattttgtcg atactacacg tagtgagggt 540
agagatcagt ttgttgattc agttgaagaa cctactagtc gtctaaattt tgacaaactt 600
gatttttcgc aagtgaaaaa tataaagagt caagctagta atgaactgac agatgagttg 660
actgacgaat tcttgaaatt gttagaagaa catttcagag atattgtctt taagaaaagt 720
tctggtgtac aattgactcc tgctgaatgg acttccgctt tggtatcgta ttttacttct 780
ttatatgaac aatcgactac aactaaatta gccgatagca aagatgcatt gaattcgttt 840
aatgttggaa aaatcaaata tgaatggcaa agaggtccag tgacaaagaa aatgacagaa 900
catttcagaa gtaagtatgg ggttgaaaat gctgagagac gtttcgctaa gaaagagtac 960
aactctattc aaaatgcttt agctgctgct gggtatgtaa gttccgaaag attggctgcg 1020
aaatggggtg ccgctcctaa taaaagaggg aaagtttctg atgctacacc gatgcataaa 1080
gctcacatga cgtttgatga acaatctgtt caattggcta gtaccgatta tgctactgat 1140
actgatacga ctagctcgaa aggagctctt catgtttcgc aagtattagg accacgtcgc 1200
ggtagaagaa attaa 1215
Claims (8)
1.一种检测LChV-1病毒的胶体金试纸条,其特征在于,所述胶体金试纸条包括PVC板,依次搭接有样品垫,结合垫,硝酸纤维素膜和吸水纸;
所述结合垫是结合有胶体金颗粒标记的兔LChV-1病毒多克隆抗体的玻璃纤维素膜;
所述硝酸纤维素膜上依据样品流动方向依次设置有检测线和质检线,所述检测线包被有LChV-1病毒重组蛋白,所述质检线包被羊抗兔IgG抗体。
2.根据权利要求1所述的胶体金试纸条,其特征在于,所述LChV-1病毒重组蛋白核苷酸序列如SEQ No.1所示。
3.根据权利要求1所述的胶体金试纸条,其特征在于,所述LChV-1病毒重组蛋白的制备方法,步骤如下:
设计带有酶切位点的特异性引物用于构建原核表达载体,引物序列如下:
LChV-1-CP-BamHI-12061-F:
AAGGATCCATGGCGAATTTAGCTTTCACGAAA;
LChV-1-CP-NotⅠ-13275-R:
TTGCGGCCGCTTAATTTCTTCTACCGCGACGTGG;
成功构建PE-HIS-LChV-1-CP原核表达载体;将PE-HIS-LChV-1-CP转入大肠杆菌表达株系的Rosetta细胞中,加IPTG诱导表达获得LChV-1病毒CP的重组蛋白;并过镍柱纯化,经得到纯化后的LChV-1病毒重组蛋白。
4.根据权利要求3所述的胶体金试纸条,其特征在于,所述IPTG的浓度为0.5mmol/L。
5.根据权利要求1所述的胶体金试纸条,其特征在于,利用纯化后的LChV-1病毒重组蛋白通过常规方法制备兔LChV-1病毒多克隆抗体。
6.根据权利要求1所述的胶体金试纸条,其特征在于,胶体金颗粒标记的兔LChV-1病毒多克隆抗体的制备,步骤如下:
调节胶体金溶液pH至7.2,将兔LChV-1病毒多克隆抗体加入胶体金溶液中,其中兔LChV-1病毒多克隆抗体的浓度为0.24mg/μL;涡旋震荡,室温放置1h后加入终浓度为1%的BSA,震荡,室温静置1h;13000rpm,4℃离心25min,弃上清,加浓度为2%的BSA(其中含0.01mol/L sodium borate)重悬沉淀,加TBS重悬沉淀,获得胶体金颗粒标记的兔LChV-1病毒多克隆抗体。
7.根据权利要求6所述的胶体金试纸条,其特征在于,所述胶体金溶液中胶体金颗粒的粒径为40nm;胶体金颗粒标记的兔LChV-1病毒多克隆抗体中多克隆抗体的浓度为2.5μL/cm。
8.如权利要求1-7任一项所述检测LChV-1病毒的胶体金试纸条的制备方法,步骤如下:
(1)将上述制备方法获得的胶体金颗粒标记的兔LChV-1病毒多克隆抗体滴加到玻璃纤维素膜上,滴加至饱和状态,室温晾干,得到结合垫;
(2)将LChV-1病毒重组蛋白和羊抗兔IgG抗体分别点涂于硝酸纤维素膜上,得到含有检测线和质检线的硝酸纤维素膜;优选的,所述检测线上重组蛋白浓度为0.24mg/ml,羊抗兔IgG抗体浓度为0.04mg/ml;
(3)在PVC板上沿样品流动方向依次搭接样品垫,结合垫,硝酸纤维素膜和吸水纸,组装得到胶体金试纸条。
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